BOR - Papers in Press, published online ahead of print
May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017608
BIOLOGY OF REPRODUCTION 69, 1085–1090 (2003)
DOI: 10.1095/biolreprod.103.017608
© 2003 by the Society for the Study of Reproduction, Inc.
Possible Expansion of "Window of Implantation" in Pseudopregnant Mice: Time of Implantation of Embryos at Different Stages of Development Transferred into the Same Recipient
Otoya Ueda2,3,
Keigo Yorozu3,
Nobuo Kamada2,3,
Kou-ichi Jishage2,3,
Yosuke Kawase2,3,
Yutaka Toyoda4, and
Hiroshi Suzuki1,3,4
Chugai Pharmaceutical Co., Ltd.,3 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan
National Research Center for Protozoan Diseases,4 Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
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ABSTRACT
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Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.
developmental biology, early development, embryo, pregnancy, uterus
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INTRODUCTION
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Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. The receptive state is defined as the "window" of limited time when the uterine environment is conducive to blastocyst acceptance and implantation. Previous studies indicated that the window of implantation is very narrow and is under strict regulation by ovarian hormones [1]. It is believed that in rodents the window of receptivity lasts for about 24 h, after which the uterus proceeds to nonreceptivity [2]. It is also widely accepted that asynchronously transferred blastocysts are held in abeyance until the uterus reaches a state of receptivity late on Day 3 of pregnancy in mice [3]. Furthermore, it is considered that transferred embryos of different ages implant in the uterus at the same time [4] or at least within several hours [5]. On the other hand, prolonged preimplantation phase experienced by asynchronously transferred embryos results in a more advanced state of embryonic development at the time of implantation, and this, in turn, sustains developmental lead and influences fetal weight [4–6]. In contrast, it has been reported that embryonic development is synchronized in the maternal uterine environment [7]. However, the previously mentioned studies did not show early implantation after asynchronous transfer. Thus, the regulation of precocious development after asynchronous transfer has not yet been established.
The present study was designed to define the time of implantation and growth synchronization of embryos of different development ages. For this purpose, embryos of two different stages were transferred separately into the oviducts of the same recipient. We then examined differences in the time of implantation and subsequent development in the uterus at different intervals after transfer until growth synchronization.
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MATERIALS AND METHODS
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ICR mice (CLEA Japan, Tokyo, Japan) were used in the present study. Donor embryos and zygotes were prepared by in vitro fertilization. The procedures used for in vitro fertilization have been described previously [8]. Zygotes up to eight-cell stage were cultured in Whitten medium [9] containing 100 µM EDTA [10] under 5% CO2 in air at 37°C. The eight-cell embryos (8C) and fertilized eggs at pronuclear stage (PN) were transferred at 54 and 6 h after insemination, respectively, into oviducts of the same recipient. Thus, PN were transferred separately to the right or left oviduct of pseudopregnant recipients on Day 0, while 8C were transferred to the other oviduct, as described previously [7, 11]. Pseudopregnancy of recipients was induced by natural mating with vasectomized male. Day 0 of pseudopregnancy was the day on which a vaginal plug was found. As controls, some of recipients received only PN or 8C embryos into both oviducts of the same mouse on Day 0 of pseudopregnancy. We chose to transfer seven PN eggs and 8C embryos to each side to provide sufficient data points. Eighty-one recipients were utilized in the experimental group. In control groups, 46 and 43 recipients received PN and 8C, respectively. The recipients were killed at 18, 42, 66, 90, and 114 h after the transfer, and both the oviducts and the uteri were flushed separately with culture medium to recover the transferred embryos. The harvested flushings were examined under an inverted microscope to assess the number and position of the recovered embryos. Only embryos of normal appearance were included in the analysis. In addition, to determine the site of implantation, 0.2 ml of 0.5% pontamine blue solution (Brilliant Blue 6B; #054-31, Nacalai Tesque, Tokyo, Japan) was injected intravenously, 15 min before killing, and blue areas along the uterus were counted. The appearance of blue bands around the uterine horns indicated that the implantation process has been initiated [12]. When the uterus was positively stained with pontamine blue, the long and short axes of the implantation site were measured by using slide calipers, and size of the implantation was calculated using the following formula: V = 4/3
a/2(b/2)2, where V = volume of implantation, a = long axis, and b = short axis. For histological examination of the implanted embryos, the recipients were killed at 90, 114, 138, 162, 186, and 210 h posttransfer. The uterus was resected and fixed with 20% neutral-buffered formalin solution and then embedded in paraffin blocks using standard procedures. It was then serially sectioned at 4-µm thickness and stained with hematoxylin-eosin. The developmental stage of implanted embryos was classified according to Theiler [13].
All mice were housed in polycarbonate cages and maintained in a specific pathogen-free environment in light-controlled (lights-on from 0500 to 1900) and air-conditioned rooms (temperature: 24 ± 1°C, humidity: 50 ± 10%). They had free access to standard laboratory chow (CE-2; CLEA Japan, Tokyo). The animals used in this study were cared and used under the Guiding Principles for the Care and Use of Research Animals promulgated by Chugai Pharmaceutical, Shizuoka, Japan.
Data presented in this study were analyzed statistically by Tukey test for nonparametric multiple comparisons. In all statistical tests, the difference was considered significant when the two-tailed P-value was < 0.05.
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RESULTS
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When the oviducts and uterine horns of 115 recipients were flushed at 18, 42, 66, 90, and 114 h posttransfer, the overall recovery rate of embryos at 42 h after transfer (37%–65%) was lower than those at other time intervals (54%–100%). Approximately 70% and 20% of the recovered embryos in each experimental group were from the oviduct at 42 and 66 h, respectively (Fig. 1A). Accordingly, the developing embryos that transferred at pronucleus and eight-cell stage seem to have migrated from the oviduct to the uterus at around 42 h after transfer. The low recovery rate of the transferred embryos at 42 h after transfer might relate to the migration; presumably, the migrating embryos were located into the uterotubal junction. Since the kinetics of migration from oviduct to the uterine horn was similar in PN and 8C embryos, migration of these embryos seems to depend on gestational age of the recipient animals rather than on the developmental stage of embryos at transfer (Fig. 1A).
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FIG. 1. Localization (A) and developmental stage (B) of embryos from the mouse genital tract at 18, 42, 66, 90, and 114 h after oviductal transfer of pronuclear eggs (PN) and eight-cell embryos (8C) on Day 0 of pseudopregnancy. PN were transferred separately into the right or left oviduct of recipients, while 8C were transferred into the other oviduct (PN-8C). As controls, PN (PN-PN) or 8C (8C-8C) were transferred into the right and left oviducts. Recipients were killed, and both oviducts and uteri were flushed separately with culture medium to recover the transferred embryos. All the recovered embryos were from oviducts of recipients in each experimental group at 18 h posttransfer. Over 60% of recovered embryos were located into uterine horns after 66 h posttransfer. The kinetics of migration from oviduct to the uterus was similar in PN and 8C embryos (A). PN eggs were recovered as morulae and blastocysts at 66 h after transfer and implanted by 90 h after transfer. However, at 66 h after transfer, 8C embryos developed to blastocysts, and some of these implanted in the uterus. Implantation of 8C embryos occurred earlier than PN (B)
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As shown in Figure 1B, preimplantation development of transferred embryos of different ages was not synchronized. PN eggs were recovered at blastocyst and morula stages at 66 h after transfer. However, 8C embryos developed to blastocyst stage, and some of these implanted in the uterus at 66 h posttransfer. Whether or not accompanied by PN egg in the reproductive tract of the recipient, implantation of 8C occurred earlier than PN. At 90 h posttransfer, pontamine blue reaction showed that 72% and 79% of embryos implanted in PN and 8C, respectively. This difference was not statistically significant (P > 0.05). Interestingly, the 48-h disparity in the developmental stage of embryos at transfer was reduced to approximately 24 h during preimplantation development.
Table 1 shows the proportion of embryos that had implanted in the uterus at 18–114 h after oviductal embryo transfer. When PN were transferred into the oviducts, pontamine blue staining was not detected in the uterus of the recipients until 90 h after transfer. In comparison, in 8C, implantation of embryos in the uterus began at 42 h posttransfer and was nearly complete by 90 h. These results indicate that the implantation of 8C precedes that of PN by over 24 h and that uterine receptivity for blastocysts is maintained for approximately 48 h when 8C and PN were transferred into the same recipient.
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TABLE 1. Rates of implantation of embryos transferred at pronuclear eggs and eight-cell stage embryos into the oviducts of recipients on Day 0 of pseudopregnancy
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To determine the time of synchronized growth of the implanted PN and 8C embryos, we estimated the volume of implanted embryo in the uterine horns using the formula for calculating the volume of an ellipsoid (Fig. 2). At 138 h posttransfer, the size of implanted PN was significantly smaller than that of 8C. At 162 and 210 h posttransfer, however, there was no significant difference in the size of implanted embryos between the two experimental groups.
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FIG. 2. Estimated volume (mm3) of implantation. Long and short axes of the implantation were measured by using slide calipers, and size of the implantation was estimated by the formula. Results are expressed as mean ± SEM, and numbers inside the bars represent number of embryos examined. *: P < 0.05, **: P < 0.01
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For further evaluation, we also performed histological analysis of implanted embryos at different intervals postimplantation. At 90 and 114 h posttransfer, there were marked differences in the development of implanted embryos between PN and 8C (Fig. 3). At 90 h posttransfer, PN were at blastocyst or implantation stage, while 8C had advanced to the egg cylinder stage. At 114 h posttransfer, PN were at the formation-of-egg-cylinder stage, while 8C matured to the differentiation-of-egg-cylinder stage. The developmental stage of PN and 8C, however, became similar, i.e., synchronized, at 162 h posttransfer, at the stage of neural plate, presomite stage (Figs. 3 and 4).
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FIG. 3. Histological examination of postimplantation development of pronuclear eggs (PN) and eight-cell embryos (8C) transferred into the oviducts of an identical recipient on Day 0 of pseudopregnancy. At 90 h after transfer, the zona-pellucida has completely disappeared in PN. The blastocyst closely adhered to the uterine epithelium. In 8C, the embryo is at the formation-of-egg-cylinder stage. Note the disappearance of the uterine epithelium in the vicinity of the embryo. At 114 h posttransfer, PN is in the formation-of-egg-cylinder stage. In 8C, the embryo is in the differentiation-of-egg-cylinder stage. The embryo has formed the proamniotic cavity. At 138 h posttransfer, PN is in the egg cylinder with formation of proamniotic cavity. In 8C, the embryo has reached the formation-of-amnion stage. Ectoplacental cavity and appearance of small lumina are seen. At 162 h posttransfer, both PN and 8C show the neural plate, presomite stage. All figures are oriented the same direction, and the right side of the plates is the antimesometrial direction. The amniotic cavities are sealed off in both embryos. Magnification, x10
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FIG. 4. Summary of development and differentiation of transferred embryos after implantation. Circles indicate individual embryos
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These results clearly indicate that the growth of transferred embryos of different ages did not synchronize during preimplantation stage but synchronized during postimplantation development. The process occurred rapidly since the differences were regulated between 138 and 162 h posttransfer (Fig. 4).
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DISCUSSION
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The established theory of blastocyst implantation, in most mammals, is that there is only a restricted period of time during the uterine cycle during which implantation can occur [14]. Failure to initiate the critical early events of implantation during this "window of receptivity" results in early failure of pregnancy. It has been considered that the mouse uterus becomes receptive for blastocyst implantation around midnight of Day 3 [15, 16] and a "cross talk" between the blastocyst and the uterus ensures initiation of implantation [1] and that the uterus becomes nonreceptive to blastocysts by the morning on Day 4 [2] or during Day 4 [17]. Furthermore, it is also widely accepted that embryos of different ages transferred to the oviduct are subsequently implanted in the uterus at the same time [4]. Doyle et al. [4] concluded that the time of implantation of two- to four-cell embryo and eight-cell morula was similar when the former were placed in one uterine horn and the latter in the other horn of recipients on Day 2 of pseudopregnancy. However, our study clearly demonstrated that the mouse uterus can potentially extend the receptivity of the endometrium for blastocyst implantation and that advanced-stage embryos implant faster than younger embryos. In fact, when eight-cell embryos and pronuclear eggs were transferred to the oviducts of mice on Day 0 of pseudopregnancy, pontamine blue reactions (i.e., number of embryos successfully implanted in the uterus) for the eight-cell embryos were detected as early as Day 2. On the other hand, uterine implantation of pronuclear eggs was only detected on Day 4 (Table 1). These results suggest that advanced-stage embryos transferred into the oviduct can implant almost 24 h earlier than native embryos. Hence, it seems that uterine receptivity can be induced in advance of the time for implantation of the native embryo. Artificial expansion of the "implantation window" might be useful for improvement of reproduction in mammals, including treatment of infertility in humans.
Previous studies have indicated that the appearance of endometrial receptivity requires priming of the endometrium by progesterone and a small amount of nidatory estrogen [1, 14]. Since serum progesterone levels significantly increase by Day 2 in both pregnant and pseudopregnant mice [18, 19], it seems that the transferred embryos can be implanted in the uterus on Day 2, as shown in the present study. Our results pose a simple question related to the results of previous studies on asynchronous embryo transfer [2, 4, 17]: Why did implantation of asynchronously transferred embryos occur at the same time, but not sequentially, in the previous studies? Although the exact mechanism was not investigated in the present study, we believe that the difference in results between the previous studies [2, 4, 17] and the present work was due to differences in the methods used for embryo transfer. While we transferred embryos to the oviduct, previous studies transferred the embryos directly to the uterus [2, 4, 17]. We speculate that the presence of the embryo into the oviduct primes the endometrium, leading to expansion of the "implantation window." In this context, recent studies have suggested that the presence of the embryo in the oviduct could result in a significant improvement in the implantation rate in mice [19]. On the other hand, when embryos of advanced stage are placed directly into the uterus, the development of the embryos might be slowed until the uterine environment is more suitable for their growth. Thus, the transferred embryos might synchronize to native embryos.
The estimated volume of implanted embryos in the uterine horns between PN and 8C shows significant difference at 186 h but not at 162 h or 210 h posttransfer. There might be a change in development at 186 h that could be related to the volume of implanted embryos. Embryonic development around 186 h posttransfer appears to correspond to first-somites stage and, further, turning-of-the-embryo stage. Turning of the embryos results in marked change of external shape in the relatively short period [13]. It seems that the change in the ratios of the "a" axis to the "b" axis (Fig. 2) could affect the estimated volume of implantation ellipsoids. Moreover, the change of "b" axis is more amplified because the "b" axis is squared in the estimation formula. However, histological analysis of the implanted embryos revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer in the neural plate, presomite stage, even though the time of implantation was accelerated in 8C compared with PN (Fig. 2). Although the mechanisms involved in the regulation of the developmental stage in sequentially implanted embryos were not identified in the present study, our results showed that the postimplantation growth of 8C was slowed by Day 6 (Figs. 3 and 4) and was synchronized to the embryonic development of PN. It is well known that aggregates of two [20, 21], three [22], four [23], and up to nine [24] embryos can grow to viable offspring of normal size. Thus, there seem to be growth control mechanisms in the embryo that can compensate for increased preimplantation size. Since aggregated embryos are not developmentally advanced when compared with control embryos of the same age, it seems that increased size cannot speed up the rate of morphogenesis [25–27]. Previous studies reported that the development of aggregates of two or four embryos is down-regulated and their size is regulated shortly after implantation at or after the time of proamniotic cavity formation, on Day 4 [25] or Day 5 [26, 27]. Thus, it appears that the mechanisms involved in the control of size become operational prior to, and are different from, those involved in synchronization of the developmental stage. It is not yet clear whether this control of proliferative activity originates in the embryo itself or is determined by the uterine environment or by "cross talk" between the embryo and uterus.
In conclusion, we have demonstrated in the present study that embryos at advanced developmental stage transferred to the oviduct can implant into the endometrium at a time prior to the interval when the uterus is prepared for the reception of native embryos. We also demonstrated that differences in morphogenesis become operational during postimplantation development, at the neural plate, presomite stage. Specifically, our results indicate that the "implantation window" can be opened in advance of acquisition of ability to implant native embryos. We have also described an experimental model that allows identification of the factor(s) responsible for the control of uterine receptivity for blastocysts: "implantation window." Future studies using this model are designed to clarify the roles of various genes such as anandamide and leukemia inhibitory factor [17] on implantation.
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ACKNOWLEDGMENTS
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The authors thank Mrs. S. Uchida for her technical assistance.
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FOOTNOTES
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1 Correspondence: Hiroshi Suzuki, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; hisuzuki{at}obihiro.ac.jp 
2 Current address: Chugai Research Institute for Medical Science, Inc., I-135, Komakado, Gotemba, Shizuoka 412-8513, Japan
Received: 27 March 2003.
First decision: 23 April 2003.
Accepted: 12 May 2003.
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INTRODUCTION
